Tuberculosis remains a major world health problem. In fact, the incidence is increasing in both the so-called developing part of the world as well as in industrialized countries like the United States of America. Recently, tuberculosis was ranked by the World Health Organization as the most frequent cause of death ascribable to a single infectious agent (Memorandum from a WHO meeting: Tuberculosis control and research strategies for the 1990s. Bulletin of the World Health Organization 70:17-21, 1992).
The means to effectively intervene transmission and thereby ultimately to get the disease under control are based on early diagnosis and treatment combined with vaccination of the populations at risk. The currently available anti-tuberculosis vaccine was developed in the beginning of this century by Calmette and Guerin and is often referred to as "the Bacille Calmette et Guerin (BCG)". The vaccine strain evolved after serial passages of a virulent isolate of M. bovis on a bile containing growth medium. The resultant strain appeared to be avirulent for humans. The nature of the loss of virulence is still not clearly understood at the molecular level. However, the BCG vaccine is estimated to be the most widely used live vaccine in the world and the remarkable low number of serious complications observed as a consequence of the use of BCG clearly demonstrate that the strain is fully attenuated (Lotte et al., Adv. Tuberc. Res. 21, 107-193 (1984)). When the reports of the first successful vaccinations were published, several laboratories and vaccine producers around the world requested the strain from Calmette and Guerin and the strain was subcultured locally under conditions which varied from one laboratory to another. This is the historical background for the occurrence of several substrains of BCG. Modern BCG producers make use of freeze-lot systems which ensure that the genetic composition of the bacteria--the product--has been conserved. Despite the widely accepted use of the BCG vaccine in many countries some countries never introduced it for use in general population vaccination programmes. This is the case in e.g. USA and Belgium. One of the reasons for these countries to be reluctant is that vaccination with BCG interferes with the use of tuberculin skin testings for diagnosing tuberculosis and for use in population surveys.
Infection of humans or susceptible animals with M. tuberculosis (or vaccination with BCG) will lead to the activation of the cellular branch of the immune system. The immunological status of a person (or animal) may therefore be monitored by analyses designed to measure the level of lymphoid cells primed against mycobacterial antigens. This may be done in vivo by measuring the "delayed type hypersensitivity (Dth) reaction" occurring 24 to 96 hours after the intracutaneous injection of mycobacterial antigen.
The product which is currently used for elicitation of Dth reactions is tuberculin--purified protein derivative (PPD). PPD consist of a crude mixture of proteins from M. tuberculosis. The proteins are recovered from synthetic medium which has supported growth of the bacteria from 5 to 6 weeks. The proteins are recovered by either ammonium sulphate or trichloric acetic acid precipitation after heat inactivation and removal of the bacterial bodies from the cultures. Contaminating lipids may be removed by ether extraction and low molecular components (&lt;10,000) are removed by ultrafiltration. However, the structural composition of virulent mycobacteria belonging to the tuberculosis complex (i.e. M. tuberculosis, M. bovis, and M. africanum) and the attenuated BCG strain is so closely related that the currently available PPD, due to cross-reactivity, will elicit a positive reaction in a large fraction of the vaccinated population. PPD is not a species specific reagent and positive reactions may also be observed when people have been exposed to or infected with other mycobacterial species.
However, other reagents have been suggested as possible reagents in a skin test for diagnosing tuberculosis. From WO 92/21697 a diagnostic skin test which comprises a 38 kDa lipoprotein or a 19 kDa from Mycobacterium tuberculosis is known. The skin test has specificity for Mycobacterium tuberculosis infections, however, the skin test cannot distinguish between patients immunised with BCG and patients suffering from tuberculosis.
Furthermore, several mycobacterial proteins, e.g. MPT70 and MPT80 from Mycobacterium tuberculosis, as well as MPB64 from Mycobacterium bovis have been shown to elicit a Dth reaction in guinea pigs sensitized with mycobacteria belonging to the tuberculosis-complex. The gene encoding MPB64 has been cloned and sequenced (Yamaguchi et al. 1989) from M. bovis BCG Tokyo.